Spatio-temporal expression profile of sirtuins during aging of the annual fish Nothobranchius furzeri

Gene Expression Patterns 33 (2019) 11–19

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Spatio-temporal expression profile of sirtuins during aging of the annual fish Nothobranchius furzeri

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Julijan Kabiljo, Christina Murko, Oliver Pusch, Gordin Zupkovitz∗ Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, 1090, Austria

A R T I C LE I N FO

A B S T R A C T

Keywords: Sirtuins Histone deacetylases Nothobranchius furzeri, epigenetics Aging

The founding member of the sirtuin family, yeast Sir2, was the first evolutionarily conserved gene to be identified as a regulator of longevity. Sirtuins constitute a protein family of metabolic sensors, translating changes in NAD + levels into adaptive responses, thereby acting as crucial regulators of the network that controls energy homeostasis and as such determines healthspan. In mammals the sirtuin family comprises seven proteins, SIRT1SIRT7, which vary in tissue specificity, subcellular localization, enzymatic activity and targets. Here, we report the identification and a detailed spatio-temporal expression profile of sirtuin genes in the short-lived fish Nothobranchius furzeri, from embryogenesis to late adulthood, mapping its entire life cycle. Database exploration of the recently published N. furzeri genome revealed eight orthologues corresponding to the seven known mammalian sirtuins, including two copies of the sirt5 gene. Phylogenetic analysis showed high cross species similarity of individual sirtuins in both their overall amino acid sequence and catalytic domain, suggesting a high degree of functional conservation. Moreover, we show that N. furzeri sirtuins exhibit ubiquitous and wide tissue distribution with a unique spatial expression pattern for each individual member of this enzyme family. Specifically, we observed a transcriptional down-regulation of several sirtuin genes with age, most significantly sirt1, sirt5a, sirt6 and sirt7 in a wide range of functionally distinct tissues. Overall, this spatio-temporal expression analysis provides the foundation for future research, both into genetic and pharmacological manipulation of this important group of enzymes in Nothobranchius furzeri, an emerging model organism for aging research.

1. Introduction Ever since their characterization as direct regulators of life span in yeast and fruitfly (Kaeberlein et al., 1999; Rogina and Helfand, 2004) the NAD+-dependent class III histone deacetylases, the so called sirtuins, continue to be at the center of interest in aging research. Sirtuins are homologues of the yeast silent information regulator Sir2 and are comprised of seven members, SIRT1-7. Based on phylogenetic analysis sirtuins have been divided into four classes (Frye, 2000). Class one consists of SIRT1, SIRT2 and SIRT3. Class II and III have one member each, SIRT4 and SIRT5, respectively, while SIRT6 and SIRT7 constitute class IV. Moreover, vertebrate sirtuins display distinct patterns of subcellular localization with a subset of sirtuins residing in predominantly nuclear (SIRT1, SIRT6, and SIRT7), cytosolic (SIRT2), or mitochondrial (SIRT3, SIRT4, and SIRT5) compartments (Houtkooper et al., 2012). Regarding their enzymatic activity most sirtuins display protein deacetylase activity. Exceptions include SIRT4, which is known only for ADP-ribosyltransferase activity, and SIRT5, which has very effective

demalonylase and desuccinylase activity and only weak deacetylase activity (Michishita et al., 2005). Together, they regulate crucial cellular and biological functions with highly divergent as well as convergent roles in maintaining metabolic homeostasis thereby determining healthspan. Each sirtuin is regulated individually in a tissue and cell specific manner. With the exception of ubiquitous Sirt6 and brain specific Sirt1, gain of sirtuin function in mammals failed to extend lifespan, (Kanfi et al., 2012; Satoh et al., 2013), although their involvement in many age associated physiological processes in vertebrates is well documented (for reviews see Finkel et al., 2009; Houtkooper et al., 2012; O'Callaghan and Vassilopoulos, 2017). Calorie restriction, which can delay the onset of age-dependent diseases such as diabetes, cardiovascular disease and cancer in rodents, and results in prolonged healthy lifespan, was directly linked to the activity of SIRT1 and SIRT6 proteins (Cohen et al., 2004; Zhang et al., 2016). Recent screenings of chemical libraries have revealed new activators of SIRT1, including resveratrol, a substance known to prolong lifespan in multiple organisms due to its ability to mimic aspects of calorie restriction (Baur

∗

Corresponding author. Center for Anatomy and Cell Biology, Medical University of Vienna Schwarzspanierstr. 17, 1090, Vienna, Austria. E-mail addresses: [email protected] (J. Kabiljo), [email protected] (C. Murko), [email protected] (O. Pusch), [email protected] (G. Zupkovitz). https://doi.org/10.1016/j.gep.2019.05.001 Received 29 January 2019; Received in revised form 24 April 2019; Accepted 6 May 2019 Available online 07 May 2019 1567-133X/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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comparison of sirtuin catalytic domains between these two species resulted in an even higher degree of similarity (Supplementary Table 2) emphasizing the conservation of class III HDACs throughout evolution.

et al., 2006; Howitz et al., 2003). Furthermore, sirtuin deficiency studies in mice, particularly knockout of Sirt1, Sirt6 and Sirt7, have additionally demonstrated the significance of these enzymes regarding promotion of healthy life and longevity (Cheng et al., 2003; McBurney et al., 2003; Mostoslavsky et al., 2006; Vakhrusheva et al., 2008). The African turquoise killifish, Nothobranchius furzeri, is an exciting new model organism for aging research. N. furzeri is a naturally short lived vertebrate that inhabits ephemeral ponds in South-East Africa and was first collected and identified as a new species in 1969 (Furzer, 1969; Jubb, 1971; Parle, 1970). Its habitat is characterized by a short rainy season followed by a long dry season. The turquoise killifish has adapted to this extreme environment by undergoing a unique annual life cycle composed of two distinct phases with opposite features, a compressed lifespan specified by rapid growth, early sexual maturation and fast aging followed by a long-term diapause state in the dry season (Blažek et al., 2013; Reichard et al., 2009). With a lifespan of 4–6 months (which is 6–10 times shorter than the lifespan of mice and zebrafish, respectively) N. furzeri is currently the shortest-lived vertebrate that can be bred in captivity (Genade et al., 2005; Terzibasi et al., 2007). Despite its short lifespan, the fish shows typical aging related phenotypes with substantial parallels to mammalian aging, such as decline in fertility, degenerative loss of skeletal muscle mass, reduced ability to learn and development of tumors (Kim et al., 2016). Moreover, it was demonstrated that calorie restriction, a resveratrol-rich diet and temperature reduction have a positive impact on N. furzeri lifespan (Terzibasi et al., 2009; Valenzano et al., 2006a, 2006b). Recently, two independent groups, (Reichwald et al., 2015; Valenzano et al., 2015), reported the successful completion of a reference genome for the shortlived turquoise killifish. In addition, methods for transgenesis (Allard et al., 2013; Hartmann and Englert, 2012; Valenzano et al., 2011) and a toolbox for precise genome-editing (Harel et al., 2015) were successfully developed. This makes N. furzeri a promising model for the exploration of vertebrate aging and aging-related diseases (Harel et al., 2015; Reichard et al., 2015; Valenzano et al., 2015). In this study we analyzed expression patterns of all NAD+-dependent histone deacetylases in seven different tissues of Nothobranchius furzeri and established a detailed age dependent spatio-temporal expression profile of all sirtuin genes, comprising the entire killifish life cycle.

2.2. Expression of sirtuins throughout the N. furzeri life cycle Studies conducted in invertebrate species such as S. cerevisiae, C. elegans and D. melanogaster have identified the sirtuin class of HDACs as one of the key regulators of lifespan (Kaeberlein et al., 1999; Rogina and Helfand, 2004; Tissenbaum and Guarente, 2001). To assess the potential change in expression levels of sirtuins in N. furzeri throughout life we analyzed five different time points broadly spanning from embryogenesis to late adulthood. The first time point (E14) chosen was 14 days post fertilization when N. furzeri reaches the black eye stage and has completed embryogenesis. The day of hatching (D01) was selected as second time point. For determination of further time points, we generated a survival curve of the GRZ strain of N. furzeri maintained in our lab from 5 weeks post hatching onwards (Fig. 2A). The survival curve obtained is comparable to those published for other N. furzeri facilities (Hartmann et al., 2009; Smith et al., 2017). Five weeks post hatching represents the age when the fish reaches sexual maturity and was taken as our third time point (W05). At the fourth time-point, week 11 (W11), the fish start showing first aging related characteristics. Obvious macroscopic aging phenotypes include progressive loss of body and tail colouration in males. At a behavioral level, older fish exhibit decreased spontaneous locomotion activity (Genade et al., 2005). At week 13 (W13), the last time-point in our analysis, which corresponds to approximately 10% survivorship, most fish show a drastic decline in their performance and display severe aging related features, such as abnormal spine curvature, loss of muscle mass and emaciation. Interestingly, a specific outward aging trait in Nothobranchius is the continuous growth of the eye (Cellerino et al., 2016). Total RNA from whole embryo/fish samples was extracted and subjected to quantitative Real Time PCR (qRT-PCR). All sirtuins were expressed at all analyzed time points, albeit with some noticeable differences (Fig. 2B). Interestingly, six of eight analyzed sirtuins, sirt1, sirt2, sirt3, sirt4, sirt5A and sirt7 showed a significant increase during the time period between hatching (D01) and reaching sexual maturity (Fig. 2B). Differential expression of these genes within this time period ranged from 10 fold for sirt5a to 1,8 fold for sirt3. Moreover, four of these genes showed gradual age-dependent reduction in their mRNA levels during adult life (Fig. 2B). Sirt5b and sirt6 did not display any significant change in transcriptional activity, neither during embryonic development nor throughout the aging process (Fig. 2B). Our data clearly demonstrate distinctive aging related trends in the expression of individual class III HDACs in whole fish extracts throughout the killifish life cycle. It furthermore suggests, that sirtuins like sirt1, sirt2, sirt4, sirt5a, sirt7 and to some extent sirt3 might be more active during the adult life of the fish, while sirt5b and sirt6 seem to execute crucial biological functions already during embryonic development. Specifically, transcript levels for sirt6 were highest at embryonic stage E14, the earliest time point examined.

2. Results 2.1. High evolutionary conservation of sirtuins To identify class III HDACs we examined the recently sequenced N. furzeri genome (Reichwald et al., 2015; Valenzano et al., 2015). Data processing retrieved eight orthologues belonging to the sirtuin gene family. We found one copy of sirt1, sirt2, sirt3, sirt4, sirt6 and sirt7 each, as well as two paralogues of sirt5. In order to gain insight into the evolutionary relation of sirtuin homologues, we performed phylogenetic analysis of seven different chordate species, human, mouse, coelacanth, medaka, zebrafish, killifish and jawless lamprey. Mouse and zebrafish were chosen as they represent standard model organisms. Medaka is the closest relative to the teleost killifish with an available genome (Reichwald et al., 2009), while lamprey represents the link between invertebrates and jawed vertebrates. Additionally, coelacanth as a living fossil bridges water and land species. The killifish sirtuins were assigned to seven well-resolved clades representing each of the sirtuin members (Fig. 1). Within every clade individual killifish sirtuins grouped together closely with the expected orthologue from other teleost fish, especially medaka (Fig. 1). Moreover, amino acid sequence similarity assessed through pairwise sequence comparison between human and N. furzeri sirtuins showed a high degree of conservation of this group of proteins. The similarity between individual human and killifish sirtuins was highest for Sirt5 with 77.5% and lowest for Sirt1 with 61.5% (Supplementary Table 2). Furthermore, the alignment and

2.3. Tissue specific expression In order to gain insight into expression of sirtuins in specific organs, we analyzed mRNA levels of each individual sirtuin in structurally and functionally distinct tissues isolated from 5 week old GRZ strain N. furzeri: brain, liver, heart, intestine, skeletal muscle, testis and kidney. After tissue extraction and RNA isolation, qRT-PCR was performed. Killifish sirt1 was expressed at comparable levels in all tissues except for the heart, where mRNA levels were significantly higher (Fig. 3). Sirt2 expression was slightly increased in skeletal muscle relative to other tissues but substantially down-regulated in testis. The main mitochondrial deacetylase sirt3 showed highest transcript levels in skeletal muscle and lowest mRNA expression in kidney and heart (Fig. 3). Sirt4, 12

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Fig. 1. Vertebrate class III HDACs are conserved throughout evolution. Phylogenetic tree of vertebrate sirtuins, generated by Maximum-Likelihood algorithm. For the analysis we used full protein sequences from the following species: human (Homo sapiens), mouse (Mus musculus), zebrafish (Danio rerio), medaka (Oryzias latipes), killifish (Nothobranchius furzeri), coelacanth (Latimeria chalumnae) and lamprey (Petromyzon marinus). Yeast Sir2 protein was used as an outgroup. The scale bar represents number of substitutions per site.

and analysis of the qRT-PCR data set (Fig. 4A) obtained from this experiment we additionally generated a heat map using Morpheus matrix visualization and analysis software (https://software.broadinstitute. org/morpheus/) (Fig. 4B, Supplementary Fig. 1). The criteria for further analysis were specified as being more than 1.5 fold difference in sirtuin expression relative to the expression levels in tissues isolated from 5 week old animals and student's t-test p values lower than 0.05. Based on age-dependent expression profiles for each specific tissue killifish sirtuins can be divided into two groups. The first group, consisting of sirt1, sirt5a, sirt5b, sirt6 and sirt7, showed a predominantly age-dependent decrease in mRNA expression, especially in muscle and intestines (Fig. 4, Supplementary Fig. 1). Two of these genes, sirt1 and sirt7, exhibited an almost identical expression profile, with light to moderate increase in brain and liver and a significant decrease in intestine and muscle (Fig. 4, Supplementary Fig. 1). Interestingly, sirt5b and its paralogue sirt5a, which both show a similar tissue distribution profile (Fig. 3), shared an age dependent expression pattern only in testis and to some extent in the heart (Fig. 4, Supplementary Fig. 1). No major age-dependent alterations in mRNA levels were observed for Sirt5b (Fig. 4, Supplementary Fig. 1). The second group, which includes sirt2, sirt3 and sirt4 displayed a predominant age dependent increase in expression, especially prominent in intestine (Fig. 4, Supplementary Fig. 1). Interestingly, sirt2 was down regulated in muscle although it showed significant age-dependent increase in expression in the majority of tissues (Fig. 4, Supplementary Fig. 1). Sirt3 was strongly up-regulated in testis and heart and down-regulated in the brain (Fig. 4, Supplementary Fig. 1) while sirt4 showed age dependent transcriptional increase in muscle, intestine and to some extent in the liver. The tissue that stood out in exhibiting the most distinctive differential expression levels of sirtuins was skeletal muscle, showing strong age depended reduction in mRNA levels in five (sirt1, sirt2, sirt5a, sirt6 and sirt7)

another mitochondrial sirtuin gene that exhibits mainly ADP-ribosylase activity was most prominently transcribed in heart and skeletal muscle (Fig. 3). Interestingly, the two paralogues of the mitochondria associated sirt5 displayed a similar spatial distribution. Both genes were highly expressed in liver, heart, intestines and muscle, but with lower mRNA levels in brain and testis (Fig. 3). The only significant difference in expression between these two paralogues manifests in the kidney (Fig. 3) where sirt5a mRNA levels were higher than those of sirt5b. Sirt6 and sirt7, both nuclear sirtuins, exhibited a similar mRNA expression profile, with higher levels in brain, liver, intestines, skeletal muscle and kidney and lower expression in testis (Fig. 3). Sirt6 also showed reduced mRNA abundance in the heart (Fig. 3). Our data show that all killifish sirtuin genes were expressed in every tissue tested, with each individual member displaying a unique spatial expression profile. 2.4. Age dependent tissue specific expression Our analyses of whole fish RNA extracts assorted the sirtuin gene family into two groups, sirt1, 2, 5a and 7, showing gradual age dependent decrease in transcriptional activity (Fig. 2B) and, on the other hand, sirt3, 4, 5b and 6, where mRNA transcription levels stayed unchanged (Fig. 2B). To gain deeper insight into the complex relationship of sirtuin gene expression and the process of aging, changes in transcriptional activity were analyzed in a tissue specific context. Therefore, in order to assess the spatio-temporal expression profile of class III HDACs during the life cycle of N. furzeri we compared mRNA levels of all killifish sirtuins from young (W05), advanced age (W11) and old fish (W13) in brain, liver, heart, intestine, muscle, testis and kidney tissues. To independently evaluate qRT-PCR data sirtuin expression was normalized to two different housekeeping genes, TATA binding protein (tbp) (Fig. 4) and ßactin (Supplementary Fig. 1). For easier visualization 13

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Fig. 2. Distinct expression patterns of killifish sirtuins throughout life. (A) Survivorship curve of the N. furzeri GRZ strain. Depicted time points indicate age of the fish at the time of sampling. The total number of fish was n = 52. (B) Quantitative Real Time PCR (qRT-PCR) of N. furzeri sirtuins sirt1, sirt2, sirt3, sirt4, sirt5a, sirt5b, sirt6 and sirt7 in whole embryo/fish cDNA samples at embryonic day 14 (E14), day of hatching (D01) and 5 (W05), 11 (W11) and 13 (W13) weeks of age. Sirtuin expression was normalized to TATA binding protein (tbp) housekeeping gene expression, with all data points for E14 set to 1. All error bars represent ± standard deviation (SD) of three biological replicas. Students unpaired t-test: ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

in both brain and liver (Fig. 4, Supplementary Fig. 1).

killifish sirtuins identified, whereas a strong increase was detected only for the sirt4 gene (Fig. 4, Supplementary Fig. 1). In contrast, kidney is the only tissue that displayed almost no change in expression of class III HDACs throughout life. Here, we detected only moderate elevation of sirt3 mRNA levels with increasing age (Fig. 4, Supplementary Fig. 1). Other tissues such as testis, intestine and heart displayed a strong aging related variance in transcript levels of sirtuins. Specifically, expression of sirt1, sirt5a and sirt7 negatively correlated with increasing age in testis and intestine, whereas sirt2, sirt3 and sirt6 showed mostly positive correlation in the heart (Fig. 4, Supplementary Fig. 1). Interestingly, sirtuin expression levels in brain and liver were only slightly affected by the age of the fish. Significant decrease in mRNA levels was detected only for sirt3 in W13 brain while levels of sirt1 and sirt7 were elevated

3. Discussion 3.1. Evolutionary relationship of sirtuins Our database analysis of the recently completed N. furzeri genome (Reichwald et al., 2015; Valenzano et al., 2015) revealed eight unique genes that belong to a group of NAD+-dependent HDACs: one copy of sirt1, sirt2, sirt3, sirt4, sirt6 and sirt7 each, as well as two copies of sirt5. The abundance of two paralogues related to a single orthologous gene in other vertebrate classes is a common occurrence for teleost fish. This is due to the teleost specific whole genome duplication (TS-WGD) that 14

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Fig. 3. Unique tissue specific expression profile of killifish sirtuins. qRT-PCRs of N. furzeri sirtuins from cDNA samples obtained from brain, liver, heart, intestines, muscle, testis and kidney of 5 weeks old fish. Sirtuin expression was normalized to tbp housekeeping gene expression, with all data points for brain set to 1. All error bars represent ± SD of three biological replicas.

decades. In invertebrates, overexpression of the Sir2 protein and calorie restriction directly resulted in prolonged lifespan (Kaeberlein et al., 1999; Rogina and Helfand, 2004; Tissenbaum and Guarente, 2001). Similar effects were also reported in killifish when subjected to calorie restriction and treatment with the Sir2 activator resveratrol (Terzibasi et al., 2009; Valenzano et al., 2006b). Also, two recent studies demonstrated that overexpression of class III HDACs directly prolongs lifespan in mammals. Whole body overexpression of Sirt6 extended lifespan in male mice, yet no similar effect was seen in female mice (Kanfi et al., 2012). The specific upregulation of Sirt1 in the murine hypothalamus as a response to dietary restriction encouraged investigators to create a transgenic mouse overexpressing Sirt1 specifically in the brain, which exhibited a phenotype of prolonged lifespan in both male and female subjects (Satoh et al., 2013). Nevertheless, most sirtuins have been identified as key regulators of diverse aging related diseases and phenotypes (Grabowska et al., 2017). Furthermore, deficiency of Sirt1, Sirt6 and Sirt7 led to premature death, accompanied by phenotypic and molecular features of accelerated aging (Cheng et al., 2003; Mostoslavsky et al., 2006; Vazquez et al., 2016). To explore killifish sirtuins in the context of aging we examined their mRNA expression levels in a number of structurally and functionally different tissues: brain, liver, heart, intestine, muscle, testis and kidney (Fig. 4, Supplementary Fig. 1). The expression levels of sirt1, along with sirt5a, sirt6 and sirt7 significantly decreased with advanced age in a number of analyzed tissues, particularly in skeletal muscle and intestines. On the contrary, genes such as sirt2, sirt4 and to a lesser extent sirt3 were mostly up-regulated (Fig. 4, Supplementary Fig. 1). Interestingly, recent results from studies conducted on the gilthead sea bream, another teleost fish, show an inverted tissue specific pattern of transcriptional deregulation of sirtuins upon dietary restriction, a known life-extending stimulus, compared to the aging-induced pattern presented here. Authors report sirt1, sirt5, sirt6 and sirt7 as being generally up-regulated upon short time fasting, while sirt2, sirt3 and sirt4 were mainly down-regulated (Simo-Mirabet et al., 2017). This implies

took place in the common ancestor of all living teleosts some 320–350 million years ago (Christoffels et al., 2004; Vandepoele et al., 2004). A similar occurrence of paralogue presence concerning sirt5 was observed in medaka (Fig. 1), a close relative of N. furzeri, and was also reported for the green spotted puffer, another teleost fish (Pereira et al., 2011). Moreover, all identified N. furzeri sirtuins displayed high cross-similarity of individual sirtuins with their respective human orthologues in both their overall amino acid sequence and catalytic domain, indicating a high degree of conservation of this group of enzymes throughout the evolution of vertebrate species. 3.2. Tissue distribution of sirtuins As previously reported for human, mouse and teleost gilthead sea bream, all N. furzeri sirtuins are present at detectable levels in all analyzed tissues of 5 week old adults (Fig. 3) (McBurney et al., 2003; Michishita et al., 2005; Simo-Mirabet et al., 2017). Interestingly however, relative tissue abundance of individual sirtuins differs from species to species. This may be explained by dynamic changes in expression of these proteins influenced by diverse impact of evolution, age and environment on the organisms analyzed. Moreover, our results demonstrate tissue specific expression profiles for each member of this group of enzymes. Additionally, we cannot exclude diverse expression patterns in specific sub organ cell types, particularly in complex organs such as the brain, intestine and testis. Given that individual sirtuins possess distinctive enzymatic activity and subcellular localization and that they are linked to different cellular mechanisms, their unique expression patterns would also imply their involvement in different physiological processes. 3.3. Age dependent expression of sirtuins Links between the process of aging and NAD+-dependent HDACs have continuously surfaced from scientific research within the past two 15

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Fig. 4. Age-dependent down-regulation of sirt1, sirt5a, sirt6 and sirt7 in muscle and intestines of N. furzeri. (A) qRT-PCR of killifish sirtuins sirt1, sirt2, sirt3, sirt4, sirt5a, sirt5b, sirt6 and sirt7 in brain, liver, heart, intestines, muscle, testis and kidney cDNA extracts at 5 (W05), 11 (W11) and 13 (W13) weeks of age. Sirtuin expression was normalized to tbp housekeeping gene expression, with all data points for W05 set to 1. Relative expression is presented on a log2 scale. All error bars represent ± SD of three biological replicas. (B) Heat-map of qRT-PCR data from (A). The mRNA expression values of sirtuin genes at W11 and W13 were normalized for W05 expression in all specified tissues. Indicated values represent only those with at least 1.5 fold difference in expression and p values lower than 0.05. Blue/ negative and red/positive values represent down-regulated and up-regulated genes, respectively. Students unpaired t-test: ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

increased DNA damage, inhibition of mitochondrial function and reduced performance (Demontis et al., 2013; Park et al., 2017). Interestingly, the sirt2 gene, which was mainly up-regulated in other N. furzeri tissues, as well as sirt1, sirt7, sirt5a and sirt6 displayed age dependent reduction in mRNA levels in skeletal muscle (Fig. 4,

that the process of aging and lifespan prolongation as a consequence of low calorie intake depend on converse expression levels of specific sirtuin genes in vertebrates. Aging of skeletal muscle tissue has been characterized by progressive loss of mass, function and regeneration potential including 16

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Supplementary Fig. 1). Our findings are in line with previous observations obtained in rodents reporting a significant age dependent decrease in Sirt7 gene expression in the rat muscle (Wronska et al., 2016). Furthermore, lifespan extending activities such as a calorically restricted diet, lead to increased levels of SIRT1 and SIRT7 in the mammalian skeletal muscle (Chen et al., 2008; Wronska et al., 2016). The role of the Sirt1 gene as a protective factor in the context of aging in mammalian skeletal muscle is evident in almost all aspects of aging muscular tissues (Zullo et al., 2018). Additionally, gain of Sirt6 function in mice led to increased insulin sensitivity in skeletal muscle and protected against high calorie diet-induced glucose imbalance (Anderson et al., 2015). Other reports have shown that SIRT1, SIRT6 and SIRT7 promote genome integrity through their involvement in DNA damage response and repair (Mostoslavsky et al., 2006; Vazquez et al., 2016; Wang et al., 2008). In N. furzeri, it was demonstrated that age dependent reduction in the number of mitochondria in skeletal muscle is associated with a decrease in ADP-stimulated and succinate-dependent respiration suggesting potential involvement of Sirt5a, which is characterized as a protein lysine demalonylease and desuccinylase (Du et al., 2011). The same study also reported that the Pgc-1α gene which is involved in the regulation of mitochondrial biogenesis and whose activity is dependent on the SIRT1 histone deacetylase (Nemoto et al., 2005), was significantly downregulated in old N. furzeri (Hartmann et al., 2011). The second most affected killifish tissue by sirtuin downregulation was the intestine, characterized by age dependent decrease in sirt1, sirt5a, sirt6 and sirt7 (Fig. 4, Supplementary Fig. 1). It was demonstrated that in mice SIRT1 can regulate intestinal inflammation during aging by altering intestinal microbiota (Wellman et al., 2017). Correspondingly, the composition of gut microbiota in N. furzeri was shown to play a key role in modulation of its lifespan (Smith et al., 2017). Moreover, SIRT1 can act protectively against age related onset of gastrointestinal tumorigenesis in rodents (Firestein et al., 2008). In addition, we observed a significant age dependent decrease in expression of sirt1, sirt7 and both paralogues of sirt5 in N. furzeri testis (Fig. 4, Supplementary Fig. 1). Besides other aging related phenotypes, turquoise killifish display an age dependent decline in fertility in both males and females (Di Cicco et al., 2011; Genade et al., 2005). Although there is little evidence on involvement of different class III HDACs in aging of mammalian reproductive organs, deficiency studies show, that loss of Sirt1 in mice is linked to a dramatic reduction of spermatids (McBurney et al., 2003). This was associated with severe morphological abnormalities, apoptotic features and increased DNA damage within the seminiferous epithelium (Bell et al., 2014; Coussens et al., 2008; McBurney et al., 2003). Remarkably, we detected only moderate to low deregulation of sirtuins in other analyzed tissues such as brain, liver and kidney (Fig. 4A and B). Importantly, published large scale expression analysis of liver and brain tissues by RNA-sequencing in the closely related N. furzeri strain MZM-04/10 revealed similar spatio-temporal sirtuin profiles, as we observed in the short-lived GRZ strain (Fig. 4, Supplementary Fig. 1). With the exception of sirt4 expression in the liver, where no age-dependent upregulation was reported, our results are consistent with the initial dataset. Of note, analysed timepoints were adapted to the longer lifespan of the MZM strain, including weeks 5, 12, 20, 27 and 39 (Baumgart et al., 2016, 2014). In accordance with our findings a study in rat including four different regions of the brain, namely frontal lobe, temporal lobe, occipital lobe and hippocampus, showed age dependent increase of Sirt1 expression in all four analyzed compartments and elevated Sirt7 transcript levels in the frontal lobe. Also, Sirt3 that was downregulated in the aging N. furzeri brain showed decreased mRNA levels in frontal lobe and hippocampus of old rat brain. Furthermore, Sirt4 and Sirt5 that were stably expressed with age in killifish displayed unchanged expression levels in the temporal and optical lobe of the aging rat brain. Sirt6 was the only member of this group of HDACs that showed a divergent transcriptional age specific regulation in all four analyzed regions compared to our study (Braidy et al., 2015).

Regarding the gene duplication of sirt5, both killifish paralogues displayed similar tissue distribution patterns with distinct spatio-temporal profiles during the process of aging, suggesting functional divergence of the two copies. However, it is yet to be defined whether this variegation is due to subfunctionalization, defined as partitioning of ancestral gene functions, or neofunctionalization, which describes assigning of a novel function to one of the duplicates, as an aftermath of the teleost genome duplication. Taken together, we show that aging is associated with the decrease in expression of sirtuin genes, most significantly sirt1, sirt5a, sirt6 and sirt7, in a wide range of functionally distinct tissues of N. furzeri. Additionally, high cross species similarity of individual sirtuins in both their overall sequence and catalytic domain, suggests a probable high degree of functional conservation further supporting the relevance of this new model organism in relation to other vertebrates, especially humans. In the last decade enormous efforts have been invested into the search for pharmacological substances with the potential of selective and specific activation or inhibition of individual sirtuins. Some of these small molecules, such as resveratrol, have already been shown to positively affect general health and longevity in multiple organisms. Future experiments involving these pharmacological compounds will serve to better understand the role that NAD+-dependent histone deacetylases play in age related pathologies and the process of aging. 4. Experimental procedures 4.1. Ethics statement All killifish experiments and procedures were performed according to the “Principles of laboratory animal care” as well as to ethical standards of the Ethics Committee of the Medical University of Vienna. Protocols were approved by the Austrian Federal Ministry of Education, Science and Research under license number BMBWF 66.009/0130-V/ 3b/2018. 4.2. Killifish care and maintenance All experiments in this report were performed using male individuals of the highly inbred N. furzeri strain GRZ (kindly donated by Dario Valenzano, Max Planck Institute for Biology of Ageing, Cologne, Germany) that were raised in the fish facility at the Medical University of Vienna. Maintenance, mating and breeding was performed as previously described (Genade et al., 2005; Zupkovitz et al., 2018a). In brief, fish were kept in a custom made overflow system, fed twice daily with frozen red mosquito larvae (Chironomidae) and maintained at 28 °C on a 12 h light/dark cycle with a fish density of one fish per 2,8 l. Selected fish were sacrificed at 5, 11 and 13 weeks of age, unless specified otherwise. Whole fish or freshly isolated dissected organs were instantly frozen in liquid nitrogen and stored at −80 °C for further analysis. 4.3. Survival curve Fish were grown in 25-L tanks containing no more than eight fish per tank. Tanks were checked daily and dead fish were instantly removed and recorded. Based on these data survival was calculated as percentage of alive fish at a certain age per total number of fish analyzed. Five weeks, the age when fish achieve their sexual maturity, was used as a starting time-point in this analysis. 4.4. Sequence analysis Full protein sequences for human (Homo sapiens), mouse (Mus musculus), zebrafish (Danio rerio), medaka (Oryzias latipes), coelacanth (Latimeria chalumnae) and lamprey (Petromyzon marinus) were obtained from the ENSEMBL database (http://www.ensembl.org). Sequences for 17

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N. furzeri were acquired from the NFINgb genome browser (http:// nfingb.leibniz-fli.de) (Reichwald et al., 2015). To artificially root the phylogenetic tree, we incorporated the yeast Sir2 protein, which was used as an outgroup. The sequence comparison was performed using the Muscle program in order to acquire high quality multiple alignments followed by filtering of low conservation parts of the alignment using the GBlocks software. The phylogenetic tree was created with the Maximum-Likelihood algorithm from the PhyML program using the LG substitution matrix. Finally, the corresponding table of both whole protein and catalytic domain sequence similarity was derived from the alignment using Geneious software. Catalytic domain sequences were identified from whole protein sequences using the InterPro database (http://www.ebi.ac.uk/interpro/). Gene identifiers from which the protein sequences were retrieved are listed in supplementary information (Supplementary Table 1).

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4.5. RNA isolation and quantitative real-time RT-PCR analysis RNA isolation from N. furzeri tissues was performed as previously described (Zupkovitz et al., 2018b). In brief, total RNA was isolated from frozen whole embryo/fish, brain, liver, heart, intestines, muscle, testis and kidney samples using TRI reagent (Sigma-Aldrich) according to the manufacturer's instructions. One μg of total RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad) and Realtime RT-PCRs were performed using the CFX384 384 Well qPCR (BioRad) and KAPA SYBR FAST Universal Master Mix (KAPA BIOSYSTEMS). Sirtuin expression was normalized to TATA binding protein (tbp) and ßactin housekeeping gene expression. Primer sequences for qRT-PCR analysis are listed in Supplementary Table 3. 4.6. Statistical analysis Evaluation of qRT-PCR experiments was performed using Microsoft Excel and GraphPad Prism software. The unpaired Student's t-test was applied to determine the significance between groups. P-values were calculated using Prism software and standard deviation (SD) is shown. *P < 0.05; **P < 0.01; ***P < 0.001. Heat map was generated using Morpheus matrix visualization and analysis software (https:// software.broadinstitute.org/morpheus/). Funding This work was in part funded by Austrian Science Fund (FWF) project J3538-B19 to C. Murko. Conflicts of interest All authors declare no conflicts of interest. Acknowledgments The authors would like to thank the Valenzano Lab, Max Plank Institute for Biology of Ageing, Cologne for kindly providing the GRZ strain and their continuous support. We are thankful to the Cellerino Lab, Pisa, for sharing their expertise and invaluable help in establishing a Nothobranchius colony in Vienna. We are thankful to Peter Auinger and Elmar Ebner for expert technical assistance in running the fish facility and for their unwavering commitment to sustain the “Austrian Notho Project”. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gep.2019.05.001. 18

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